During the last decades much progress has been made to improve the efficacy of diagnostic and therapeutic drugs. Major achievements have been made in acute diseases. Today, acute diseases such as infectious diseases, acute thrombosis or acute dysregulation of blood pressure can be treated with high efficacy. Most drug treatments for acute diseases do not seriously affect healthy tissues and organs. Because of the short period of drug treatment healthy tissues and organs can sufficiently recover from unwanted drug effects. In contrast to the short lasting drug treatment of acute diseases which is usually accompanied with a short period of drug exposure, treatment of chronic diseases is indispensible associated with a long lasting exposure of the human body to the applied drugs. The long lasting exposure of the drug, however, often harms healthy tissues and organs.
Two different strategies were followed during the last decades to avoid severe unwanted drug effects on healthy tissues and organs. On the one hand, drug research was focused on new drug targets that promised a disease-specific expression of the target mechanism. With respect to the discovery of signal transduction mechanisms in proliferating and activated cells, numerous new targets have been identified. However, with few exceptions therapeutic attack of the majority of newly discovered drug targets did not improve therapeutic outcome. On the other hand side, much effort has been devoted to improve the bioavailability of clinically established therapeutic drugs. In order to improve bioavailability drug research focused on the chemical modification or pharmaceutical formulations of the therapeutic or diagnostic effector molecules.
Over the past three decades the use of targeted effector conjugates has been well established. In particular, molecules which can induce diagnostic or therapeutic effects are linked to a carrier molecule with targeting properties. Due to the high binding affinity of immunoglobulins, protein antibodies or antibody fragments are frequently used as carrier molecules for targeted delivery. With respect to the treatment of neoplastic diseases, antibodies may carry toxins or chemotherapeutic agents to the tumor. Because of the strong binding of the antibody-effector conjugates to certain target molecule of tumors, a significantly higher concentration of the effector in the tumor environment is achieved. Meanwhile, antibody-effector conjugates have proven effective in a series of experimental and clinical tumors. Another advantage of the targeted delivery of effector molecules is the reduction of unwanted effects of the effector molecule. In detail, the majority of drug molecules which are not linked to the carrier with targeting properties do not reach the site of the disease and are only applied into the human body in order to achieve necessary drug concentration in the blood. Therefore, the utmost portion of the applied drug is eliminated from blood circulation without reaching the site of the disease. For example, malignant solid tumors which can be regarded as a chronic disease have a size of 1 to 10 gram at the time point of diagnosis and treatment, therefore, represent 0.01 to 0.001% of the human body. This ratio illustrates that drug treatment can be significantly optimized by directing the applied drug to the disease, and, therefore, enable reduction of the applied dose.
However, despite of remarkable progress in the treatment of acute diseases, the majority of treatments fail to achieve cure from the chronic disease. In contrast to acute diseases, most chronic diseases can only be treated if disease-related signal transduction and gene transcription can be selectively targeted. In order to achieve this goal, therapeutic drugs have to sufficiently permeate the cell membrane and to accumulate within the target cell of the disease. Because of the ubiquitous expression of the key target molecules of gene transcription and protein synthesis, future therapeutic drugs have to demonstrate selective uptake at the site of the disease. The latter characteristic of future therapeutic drugs is of crucial importance because binding to and inhibition of key regulators of gene transcription and protein synthesis outside the disease process may harm the human body.
Results from scientific investigations provided evidence for a role of more than 500 factors in gene transcription and protein synthesis. However, among the different factors, NF-kappaB and AP-1 are crucial and have already been established as therapeutic targets (Letoha et al., Mol. Pharmacol. 69: 2027, 2006; Sliva et al., Curr Cancer Drug Targets. 4: 327, 2004) These two regulators of gene transcription play a central role in activation and proliferation of cells and are the downstream signal of different signaling cascades. Both NF-kappaB and AP-1 are located in the cytoplasm and nuclei of cells. With respect to therapeutic targeting of NF-kappaB and AP-1, drugs have to fulfill two important prerequisites. First, a therapeutic drug has to permeate the cell membrane in sufficient amount and to accumulate within the cytoplasm. Second, the therapeutic drug has to discriminate between cells in healthy organs or tissues and cells in the disease process. The latter aspect is of great significance because NF-kappaB and AP-1 are expressed in every cell of the human body and an inhibition of these two disease targets may significantly harm sensitive body functions.
NF-kappaB (nuclear factor kappa-light-chain-enhancer of activated B cells) is a protein complex that controls the transcription of DNA. NF-kappaB is found in almost all animal cell types and is involved in cellular responses to stimuli such as stress, cytokines, free radicals, ultraviolet irradiation, oxidized LDL, and bacterial or viral antigens. NF-kappaB plays a key role in regulating the immune response to infection. Conversely, incorrect regulation of NF-kappaB has been linked to cancer, inflammatory and autoimmune diseases, septic shock, viral infection, and improper immune development. NF-kappaB has also been implicated in processes of synaptic plasticity and memory (Baud et al., Nat Drug Discov. 8:33, 2009).
NF-kappaB is widely used by eukaryotic cells as a regulator of genes that control cell proliferation and cell survival. As such, many different types of human tumors have misregulated NF-kappaB: that is, NF-kappaB is constitutively active. Active NF-kappaB turns on the expression of genes that keep the cell proliferating and protect the cell from conditions that would otherwise cause it to die via apoptosis. Defects in NF-kappaB result in increased susceptibility to apoptosis leading to increased cell death. Because NF-kappaB controls many genes involved in inflammation, it is not surprising that NF-kappaB is found to be chronically active in many inflammatory diseases, such as inflammatory bowel disease, arthritis, sepsis, gastritis, asthma, among others. Many natural products (including anti-oxidants) that have been promoted to have anti-cancer and anti-inflammatory activity have also been shown to inhibit NF-kappaB (Kaur et al., Curr Cancer Drug Targets 7: 355, 2007).
Activator protein 1 (AP-1) is a transcription factor which is a heterodimeric protein composed of proteins belonging to the c-Fos, c-Jun, ATF and JDP families. It regulates gene expression in response to a variety of stimuli, including cytokines, growth factors, stress, and bacterial and viral infections. AP-1 in turn controls a number of cellular processes including differentiation, proliferation, and apoptosis (Vesely et al., Mutat Res. 682: 7, 2009).
Activation of NF-kappaB and AP-1 results into transcription of genes encoding for numerous signaling molecules involved in tumor growth, apoptosis, inflammation, autoimmune disease and fibrosis. Cytokines such as interleukin-1, interleukin-6, TNF-alpha or growth factors like TGF-beta (TGF-β) represent the most important downstream signals of NF-kappaB and AP-1 activation. In particular, transforming growth factor beta (TGF-beta) is a highly pleiotropic cytokine that controls many aspects of cellular function, including cellular proliferation, differentiation, migration, apoptosis, adhesion, angiogenesis, immune surveillance, and survival and, therefore, represents an important target for therapeutic drugs (Jakowlew, Cancer Metastasis Rev 2006; 25:435-57). TGF-beta is produced by many cell types, is always present in the plasma (in its latent form) and permeates all organs, binding to matrix components and creating a reservoir of this immunosuppressive molecule. Anyway, it is overproduced in many pathological conditions. This includes pulmonary fibrosis, glomerulosclerosis, renal interstitial fibrosis, cirrhosis, Crohn's disease, cardiomyopathy, scleroderma and chronic graft-versus-host disease (Prud'homme et al., Lab Invest 2007; 87:1077-91). In neoplastic disease, TGF-beta suppresses the progression of early lesions, but later this effect is lost and cancer cells produce TGF-beta, which then promotes metastasis. This cytokine also contributes to the formation of the tumor stroma, angiogenesis and immunosuppression (Jakowlew, Cancer Metastasis Rev 2006; 25:435-57). In view of this, several approaches are being studied to inhibit TGF-beta activity, including neutralizing antibodies, soluble receptors, receptor kinase antagonist drugs, and antisense reagents. The benefits of new therapies targeting TGF-beta are under intense investigation (Prud'homme, Lab Invest 2007; 87:1077-91).
For a therapeutic intervention all autoimmune diseases are considered where the pathological process is characterized by a defect and unregulated interaction of cellular and non-cellular components of the immune system such as coeliac disease, diabetes mellitus type 1 (IDDM), systemic lupus erythematosus (SLE), Sjögren's syndrome, Churg-Strauss Syndrome, multiple sclerosis (MS), Hashimoto's thyroiditis, Graves' disease, idiopathic thrombocytopenic purpura, Addisons disease, anemia, ankylosing spondylitis, osteoarthritis, Behcets Syndrome, Canker Sores, chronic fatigue, chronic obstructive pulmonary disease (COPD), Crohns disease, Cushings disease, dermatitis herpetiformis, dermatomyositis, eczema, fibromyalgia, hair loss, hepatitis, hypothyroidism, lichen planus, Meniere's Disease, myasthenia, Reiters Syndrome, sarcoidosis, scleroderma, sepsis, Sjogrens Syndrome, sun poisoning, SIRS (systemic inflammatory response syndrome) and uveitis (Masters et al, Annu Rev Immunol 2009; 27:621-68).
In human cancers, TGF-beta is produced by activation of NF-kappaB or AP1 and promotes tumorigenesis through both decreased TGF-beta signaling during early tumorigenesis and increased TGF-beta signaling in advanced, progressive disease. There is evidence that TGF-beta regulates the cell-cycle activity of tumor cells leading to a control of tumor cell proliferation. While the growth of normal cells and differentiated tumor cells is blocked by TGF-beta, the growth of undifferentiated tumor cells is stimulated. The stimulatory action of TGF-beta in undifferentiated tumor cells is due to a mutated signaling pathway. Despite the effect of TGF-beta on the growth of primary tumor cells, TGF-beta is one of the most potent regulators of the tumor metastasis through a stimulation of the tumor cell extravasation. An effect on the tumor angiogenesis is another mechanism of TGF-beta to stimulate tumor growth and metastasis (Tian et al., Future Oncol 2009; 5:259-71). Elevated levels of TGF-beta were found in a number of tumors as acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphoma, astrocytoma, basal cell carcinoma, skin cancer (nonmelanoma), bile duct cancer, bladder cancer, bone cancer, fibrous histiocytoma, brain tumor, breast cancer, bronchial tumors, Burkitt lymphoma, carcinoid tumor, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous T-Cell lymphoma, Mycosis Fungoides, embryonal tumors, esophageal cancer, eye Cancer, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, germ cell tumor, glioma, hairy cell leukemia, head and neck cancer, hepatocellular (liver) cancer, Hodgkin lymphoma, islet cell tumors, kidney (renal cell) cancer, laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, myelodysplastic syndromes, nasopharyngeal cancer, ovarian cancer, pancreatic cancer, parathyroid cancer, prostate cancer, benign prostate hyperplasie, rectal cancer, sarcoma, stomach cancer, thyroid cancer, vaginal cancer (Jones et al., Expert Opin Ther Targets 2009; 13:227-34).
A critical role for TGF-beta was also corroborated in diseases of the cardiovascular system. Very similar to the mechanism of TGF-beta induction in tumors, a major stimuli of TGF-beta synthesis in cardiovascular disease is activation of NF-kappaB, too (Frangogiannis, Pharmacol Res 2008; 58:88). TGF-beta has been implicated in many cardiovascular disorders such as stroke reperfusion, ischemia, heart attack, myocarditis, endocarditis, myocardial insufficiency (Goumans et al, Trends Cardiovasc 2008; 18:293-8). TGF-beta has important roles in the development of the neointima and constrictive remodeling associated with angioplasty. In atherosclerosis its actions are yet to be fully elucidated but its ability to control the immune system has profound effects on lesion development, particularly by influencing the types of lesions that develop. TGF-beta can also induce arteriogenesis and markedly influences angiogenic processes, possessing both pro- and anti-angiogenic effects (Galinka et al, Annu Rev Immunol 2009; 27:165-97). It is also a major contributor to the development of various cardiovascular fibrotic disorders including those in the vasculature, heart and kidney. TGF-beta was also shown to play an important role in the development and progression of fibrosis. Fibrosis is the formation or development of excess fibrous connective tissue in an organ or tissue as a reparative or reactive process, as opposed to a formation of fibrous tissue as a normal constituent of an organ or tissue. Examples are cystic fibrosis of the pancreas and lungs, injection fibrosis, which can occur as a complication of intramuscular injections, endomyocardial fibrosis, idiopathic pulmonary fibrosis of the lung, mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, progressive massive fibrosis, a complication of coal workers' pneumoconiosis, nephrogenic systemic fibrosis (Pohlers et al., Biochim Biophys Acta, 2009, 1792, 746-756).
Currently available anti-TGF-beta therapeutic drugs exert several disadvantages. The significant disadvantage of established drugs for treatment of autoimmune disease is their small therapeutic window. Repeated applications usually lead to adverse drug effects and severe organ damages. Cardiotoxicity, nephrotoxicity and hepatitis are common side effects of clinically available drugs for treatment of autoimmune disease (Cohen, International Journal of Clinical Practice 2007; 1922-1930). In the clinical setting, most established drugs are intermittently applied to avoid irreversible toxicity. However, intermittent treatment schedules increase the risk of disease progression. For these reasons, a significant need for more efficient and well tolerated drugs for treatment of TGF-beta related diseases exists.
It is known that TGF-beta can be inhibited by several approaches leading to an inhibition of the receptor signaling. However, these approaches are hampered by a limited efficacy and lack of tolerability in vivo. The synthesis of antisense oligonucleotides to block TGF-beta was described (Flanders, Clinical Medicine & Research 2003, 1, 13-20). Antisense oligonucleotides can diminish the synthesis of the TGF-beta protein. However, this approach does often lead to an incomplete inhibition of TGF-beta synthesis. Another disadvantage of antisense oligonucleotides is the low amount of drug accumulated at the site of disease. Small molecule inhibitors (SMIs) of the TGF-beta receptor are also known (Hjelmeland et al., Mol Cancer Ther 2004; 3: 737-745). These molecules are often orally available but lack sufficient tolerability and safety. The toxic side effects of known SMI's of the TGF-beta receptor are due to a lack of specificity. The known compound do not only inhibit signaling of the TGF-beta receptor but also many other receptors with structural similarities. Antibodies that bind TGF-beta or block TGF-beta binding to its receptors are also known. These molecules show sufficient accumulation at the site of the disease and do block signaling over a long period (Saunier et al, Curr Cancer Drug Targets 2006; 6:565-78). However, antibodies bear several disadvantages which do limit their therapeutic application. First, antibodies interfering with TGF-beta may exert unwanted side effects due to activation of the immune system by parts of the antibodies carrying binding sites to components of the immune system. This activation of the immune system can lead to toxicity of the treatment. Another disadvantage may be the production of neutralizing antibodies. The onset of neutralizing antibodies is frequently observed after multiple applications. In case of neutralizing antibodies the efficacy of the treatment is decreased.
Because of the limitation of the known treatments, novel approaches to treat diseases related to activated NF-kappaB and AP-1 and elevated synthesis of TGF-beta are required. The ultimate goal of a novel therapeutic approach is high efficacy and good tolerability. Therefore, it is an objective of the present invention to provide compounds and compound classes which are easy to synthesize and which are suitable for the treatment of disease associated with activation of NF-kappaB or AP-1 and elevated synthesis of cytokines such as TGF-beta. According to the invention, it was surprisingly found that polyanionic multivalent macromolecules represent a novel class of therapeutic molecules that selectively deliver effector molecules into the cytoplasm and nuclei of proliferating and activated cells.
The invention proposes the use of polyanionic macromolecules based on the multivalent assembly of a plurality of sulfate groups on a dendritic branched macromolecular carrier for intracellular delivery of diagnostic or therapeutic effector molecules. More specifically, the invention comprises the use of sulfated polyols with hyperbranched structure to which diagnostic or therapeutic effector molecules are covalently attached as drugs to treat diseases related to activated NF-kappaB and AP-1 and elevated synthesis of TGF-beta.